Breeding plants

ABSTRACT

A process for breeding plants which comprises growing plants of a species in an array of containers charged with growing medium of uniform characteristics in an environment of controlled climatic conditions with controlled supply of nutrients and feed water and changing the positions of the containers within the environment as required to ensure at least substantially uniform exposure of all plants in the containers to conditions in the environment. A process for the breeding of open pollinating plants in a greenhouse environment is also provided.

This application is the U.S. national phase of international applicationPCT/EP2004/050085, filed 5 Feb. 2004, which designated the U.S. andclaims priority of EP 03075350.3, filed 5 Feb. 2003, and EP 03103779.9,filed 13 Oct. 2003, the entire contents of each of which are herebyincorporated by reference.

This invention relates to breeding plants and is especially concernedwith the conditions under which plants are grown during the breedingprocess.

Plant breeding starts in essence with the creation of genotypicvariation among plants of a given species. The creation of genotypicvariation relies on the production of genetic alterations that can beobtained by techniques including recombination through classicalcrossing, chemical mutagenesis, radiation-induced mutation, somatichybridisation, inter-specific crossing and genetic engineering.Following the creation of genotypic variation, selection of thosegenotypes having the most desirable agronomic phenotypes is performed.

In plant breeding, the process of selecting those genotypes having themost desirable agronomic phenotypes is typically carried out in fieldscomposed of several plots, each plot typically consisting of at leastseveral square meters or of sufficient dimensions to grow at leastseveral tens of plants. The assessment of phenotypes in a statisticallysound manner requires that each genotype be grown and its phenotypeassessed in several different plots, typically no less than three. Evenbefore the field experiments may begin, the plant breeder requiresenough seeds to grow the required number of plants for the experiment.Seeds of a given genotype are often only available in small quantitiesand therefore the generation of greater seed numbers is often requiredbefore the experiments may begin. Seed generation is typically carriedout by the growing of sometimes multiple generations of plants in orderto obtain the necessary seed quantities to conduct a field scaleexperiment.

For breeding to be successful, a sufficient number of geneticalterations has to be examined in order to identify the few amongst manythat are of agronomic relevance. The procedure for selection ofgenotypes has to be sufficiently discriminative for detecting phenotypicdifferences between the different genotypes and requires as a basis aset of parameters that is sufficiently detailed as to adequatelydescribe the observed phenotypes.

The phenotype (based for example on observations of growth habit, yieldpotential and resistance to stresses) is the result of contribution fromthe genotype itself (genotype-associated phenotype) and from theenvironment (environment-associated phenotype). Theenvironment-associated phenotype is influenced by variations in thegrowth environment caused by variations in, for example, temperature,humidity, light, nutrient and supply. An important factor that obscuresphenotype-driven selection of desired genotypes is variations in theenvironment-associated phenotype component. When phenotype-basedbreeding is performed in the field, most of the environmental variationcomes from non-uniformity of the soil in which plants are grown. Thesoil composition, the physical properties of soil, the availability ofnutrients, water and the presence of microbial soil inhabitants can varyover short distances in natural soils. One way to avoid variations dueto the heterogeneity of soil is to grow plants in containers (pots,trays, or the like) containing a better defined substrate, such aspotting soil, vermiculite, or rock wool. Growth of plants on a definedsubstrate is usually done in a greenhouse, so that the amount of waterand nutrients given to the plants can be controlled. Growing plantsunder these conditions significantly reduces environmental variation.However, even when plants are grown in a greenhouse equipped withsystems for climate control (heating, cooling, aeration, humiditycontrol systems) and nutrient/water delivery, environmental conditionsstill vary with geometrical locations within the greenhouse. Thisenvironmental variation can be due to differences in distance betweenplants and the proximity or otherwise to devices used for climatecontrol and nutrient/water delivery, for example heating elements,cooling elements, windows, doors, misting devices, ventilators, waterinlets and water outlets.

Plants grown in greenhouses are amenable to automated handlings becausethey are usually grown in pots that can be easily transported to andfrom automation devices.

When breeding open pollinating plants, such as corn, in a greenhouseenvironment, it is important to restrict the flow of pollen within theenvironment. Failure to do so could, undesirably, lead to a mixing ofthe various genetic characteristics present within the environment.Conventional means for the growing of corn, an open pollinating plant,in a greenhouse environment deal with the necessity to restrict pollenflow by covering the female parts of the plant. In the case of corn,each female organ (ear with extending silks) is covered with a (paper)bag so as to prevent the contacting of pollen with these parts.Pollination is then carried out in a controlled manner by removing thebags, so as to expose the female parts, and then shaking the tassel(male organs of a corn plant) in the neighbourhood of the female organs.This process generates considerable variability in pollinationefficiency, not only due to the variation in quantity and quality of thepollen applied, but also due to the difficulty associated with timingthe pollination event given the short window available in which thefemale organs are in an optimal state for pollination. There aretherefore major problems associated with conventional methods for thebreeding of open pollinating plants in a greenhouse environment.

It is one object of the present invention to provide an improved processfor breeding plants.

It is another object of the present invention to provide an improvedprocess for breeding plants in which plant breeding may be conductedusing smaller seed quantities than in conventional breeding processes.

It is another object of the present invention to overcome some of theaforementioned problems and to provide an improved process for thebreeding of open pollinating plants in a greenhouse environment.

We have now found that one may reduce significantly the influence ofenvironment-associated phenotype on phenotyping results by changing thelocation of growing plants in a greenhouse. The influence ofenvironment-associated phenotype on phenotyping results may be furtherreduced by growing the plants on a growth medium of uniformcharacteristics in an environment of controlled climatic conditions witha controlled supply of nutrients and feed water.

The invention therefore provides in one of its aspects a process forbreeding plants which comprises growing plants of a species in an arrayof containers charged with growing medium of uniform characteristics inan environment of controlled climatic conditions with controlled supplyof nutrients and feed water and changing the positions of the containerswithin the environment as required to ensure at least substantiallyuniform exposure of all plants in the containers to conditions in theenvironment. A process according to invention preferably comprises thefurther step of identifying phenotype characteristics such as, growth,yield and tolerance to abiotic stress of the plants at intervals in thegrowing cycle. For example, plants may be evaluated visually or by useof appropriate equipment. If desired, algorithms may be used to selectand evaluate the information and the results statistically analysed toidentify trait leads. Preferably also, plants are selected for furtherbreeding or for commercial use by comparing the phenotypecharacteristics of the plants.

The word “comprising” where used herein is intended to encompass thenotion of “including” and the notion of “consisting essentially of”.

In a process according to invention, the positions of the containers maybe changed continuously or at intervals. In one preferred process theyare changed to an extent and at intervals pre-set by an operator inaccordance with observation of growth characteristics of the plants. Thepositions of the containers within the environment may be changed atintervals of up to two weeks, preferably from six hours to two weeksmore preferably at intervals from one day to one week. Preferably thepositions of the containers within the environment are changedautomatically. One preferred apparatus supports an array of containersin the form of pots containing plants. Preferably there is one plantgrowing in each pot, although plants can be grown with severalindividuals in a large pot or in trays or in a tray consisting ofphysically connected pots.

Apparatus suitable for use in a process according to the inventionpreferably comprises transporter means upon which the containers aresupported in a horizontally disposed uniform array. Preferably thetransporter means comprises a plurality of co-extensive storagetransporters each comprising a “U” shaped gutter equipped with a beltlying flat on the bottom of the gutter, and transfer conveyor means.Preferably the belts are moved in the gutters by means of motors such aselectric motors. In one preferred apparatus hereinafter described,shuttle robots are provided which can position themselves in front of aparticular gutter, and a motor in the robot can be activated to pull thebelt in the gutter back or forth, thus allowing the transport ofcontainers in and out of the gutters. The ends of the gutterscommunicate with the transfer conveyor, so that containers may betransported back and forth from gutter to gutter and back and forth tospecific areas in the array. Preferably, the motors are caused to movethe containers continuously or at time intervals for example intervalsfrom six hours to two weeks more preferably at intervals of one day toone week. Thus, a storage transporter may be moved by motor means tomove the row of containers supported on that storage transporter towardsa first transfer station at which an endmost container of the row istransferred to the transfer conveyor means, the transfer conveyor meansmay be operated to move a container supported on it to a second transferstation and motor means of a second storage transporter may be operatedto collect the container.

In one preferred arrangement, the transfer conveyor means is arranged sothat the container is carried in a linear path from the first mentionedstorage transporter to the second storage transporter. In anotherarrangement, the transfer conveyor means is arranged so that thecontainer is carried in a closed path from the first mentioned storagetransporter to the second storage transporter; for example a rotarytable may be employed and if so, several containers may be loaded to itand removed therefrom in random order for sending to the second storagetransporter.

In a process according to the invention the containers are positioned asclosely together as practicable bearing in mind the volume occupied bythe plant or plants in the container. A process according to theinvention has various advantages. It enables effective dampening ofenvironmental variations that influence the phenotypes of plants andtherefore interfere with the selection of desired genotype-associatedphenotypes. By changing the location of plants during their life cyclethey are exposed to slightly different environmental settings at eachlocation. When the displacement of all the plants in the breedingpopulation occurs at a sufficiently high frequency (e.g. once per day oronce per week) then the spatial effects will be randomized over thepopulation. The dampening of environmental contribution to the phenotypeof plants during breeding enables desired genotypes to be selected morereliably. The step of identifying the phenotype (be it a characteristicsuch as growth, yield or stress tolerance) is facilitated by thechanging of the location of plants. For example, where the plantphenotype is evaluated by means of appropriate equipment, the plants maybe moved at appropriate intervals to the station where the equipment forevaluation is located. Also, enhanced uniformity of the growing plantsallows use of a smaller plant population for study, which in returnreduces costs. This allows for the breeding of various agronomiccharacteristics, such as yield, using a smaller plant population(typically on a green-house scale) compared to conventional methodsrequiring much larger plant populations (typically on a field scale) inorder to accurately assess the phenotypic variation associated withdifferent genotypes. Also, the process gives a more efficient plantbreeding because, given a fixed number of genetic alterations studied,fixed level of required discriminative power, the size of the populationrepresenting a certain genetic alteration can be reduced resulting inthe capacity to reliably establish phenotypes under conditions oflimited availability of seeds (as is often the case in the generationfollowing the creation of genetic alterations), as such, omitting theneed for extensive seed propagation and consequent loss of time.

The advantage of being able to conduct a breeding process with smallerseed quantities than would otherwise be possible is particularlyrelevant in the case of breeding of transgenic plants for variousdesirable phenotypic characteristics (traits).

The breeding of transgenic plants involves the introduction of at leastone nucleic acid into a single plant. Different plants may have the sameor different nucleic acids introduced therein. Plants are then grown andevaluated to identify plants having desirable traits. Plants having suchdesirable traits may be further evaluated in a field environment or maybe backcrossed with different varieties of inbred lines of the same cropspecies or may be used to generate or test hybrids or may be used forthe production of seed, possibly for commercial use.

Advantageously, any plant may be subject to the breeding process of theinvention. According to one aspect of the invention, the plant subjectto the breeding process is a self-pollinating plant, such as rice. Aself-pollinating plant is one in which, under normal conditions, thefemale organs of any one given plant species is pollinated by pollenproduced in the male organs of that same plant species.

According to another aspect of the invention, the plant subject to thebreeding process is an open pollinating plant species, such as corn. Anopen pollinating plant species is a plant which is substantially anon-self pollinating plant species.

The present invention also provides a process for the breeding of openpollinating plant species in a greenhouse environment This process,which is applicable to open pollinating plants, is preferably used inconjunction with the process for breeding plants described hereinabove.Advantageously, the process for the breeding of open pollinating plantsremoves the variability in pollination efficiency associated withconventional methods for the breeding of open pollinating plants, suchas corn, in a greenhouse environment. A further advantage of thisprocess is that the problem of the mixing of genotypes, associated withconventional methods, is avoided.

Therefore according to the present invention, there is provided aprocess for the breeding of open pollinating plants in a greenhouseenvironment, comprising the steps of:

-   -   (i) providing a male sterile open pollinating plant;    -   (ii) contacting the male sterile plant with pollen; and        optionally    -   (iii) selecting plants having a desired phenotype

The aforementioned process is particularly applicable to the breeding ofopen pollinating plants, in particular corn. In the process of theinvention, pollination is made more efficient, and the mixing ofgenotypes avoided, by rendering the plants subject to the breedingprocess male sterile. This substantially eliminates any self pollinationwhich may occur and further substantially eliminates the mixing ofgenotypes within the population of plants. Methods for rendering plantsmale sterile are well known in the art and include chemical ormechanical techniques. An example of a mechanical technique forrendering plants male sterile simply involves the physical removal ofmale parts (emasculation). An example of a chemical technique forrendering plants male sterile involves the use of chemical hybridisingagents or CHAs.

The process comprises the further step of contacting the male sterileplants with pollen. This is carried out by the introduction of malesterile plants to a pollination chamber in which male sterile plants aregrown for a period ranging from about 2 days to about 3 weeks,preferably from between about 3 days and about 10 days. The introductionof male sterile plants into the pollination chamber coincides with aperiod of female fertility. A person skilled in the art will readilyrecognise the period of female fertility and thus will be well aware ofthe point in time for the introduction of the male sterile plants to thepollination chamber and the time required for maintenance of the malesterile plants in the pollination chamber. The pollination chambercomprises a number of pollinator (pollen shedding) plants. Male sterileplants, upon introduction into the pollination chamber, are grown for aperiod of time with pollinator plants of a single variety of the samespecies, most preferably of the same variety as the male sterile plants.In order to optimise contacting of pollen with the male sterile plants,pollinator plants are interspersed between male sterile plants. In onearrangement, a row of pollinator plants is provided followed by a row ofmale sterile plants followed by another row of pollinator plants and soon. Alternatively, one pollinator plant may be placed next to a malesterile plant so as to fill all rows in a crisscross formation. Thepollination chamber may be arranged such that it is physically orspatially separate from the location where the male sterile plants growin times outside of female fertility. The pollination chamber isequipped with a controlled environment (climatic or otherwise) optimalfor pollination and depending upon the requirements of thecross-pollinating species to which the process is applied. By way ofexample, for corn, the controlled climatic conditions comprise atemperature of between about 20° C. and about 35° C., preferably betweenabout 24° C. and about 30° C. and a relative humidity of between about70% and about 90%. Furthermore, since corn is pollinated principallywith the aid of wind, the pollination chamber is equipped with means topromote circulation of air and therefore pollen throughout the chamber.Fertilisation of plants is further aided by provision of a pollinationchamber of such dimensions so as to create a sufficient concentration ofpollen to promote fertilisation.

The process optionally comprises the further step of selection of plantsfor any given characteristic. This may be selection based on thepresence of a particular phenotype or may be selection of plants havingappropriate dimensions to fit, say, the transporter means or to be of anappropriate height to be suitable for a greenhouse environment, forexample taking into account the proximity of the plants to the lightsetc. With regard to corn, there are particular advantages associatedwith the use of plants which are relatively small or short and whichhave a comparatively short cycle time, preferably about four months orless. These plants may be different varieties, hybrids, an inbred or apopulation, such as Gaspe or any inbred line derived from Gaspe e.g.through a number of generations of selfing.

In order that the invention may become more clear there now follows adescription to be read with the accompanying drawings of apparatus foruse in a preferred process according to the invention selected fordescription to illustrate the invention by way of example. In thedrawings,

FIG. 1 is a schematic representation of transporter means of theillustrative apparatus; and

FIG. 2 is a view in perspective of a channel member and conveyor belt ofthe transporter means supporting a plant pot.

The illustrative apparatus is suitable for use in conjunction with aplurality of containers in the form of plant pots (10) (FIG. 2) in whichone or more plants is growing in a medium selected for the purpose.

The apparatus comprises transporter means (20) by which the pots aresupported and moved as desired. The transporter means (20) comprises aplurality of co-extensive storage transporters (22) each providingsupport for a row of several pots, the storage transporters beingdisposed adjacent one another to support rows of pots in a horizontallydisposed array. Each storage transporter (22) comprises a channel memberprovided by a rigid “U”-shaped gutter (24) secured in parallel relationnext to adjacent gutters. An endless belt (26) operates within eachgutter (FIG. 2) and is located with an upper surface lying in the gutterand arranged to be drawn along it. Each belt (26) supports a row ofclosely spaced pots (10). The gutters (24) are situated with their endportions proximate to a belt conveyor (30) of transfer conveyor means(28) located transversely to the gutters (24).

Electrically operated shuttle robots (32, 34) are employed to actuatemovement of the belts (26) in the gutters (24). The movement causes thepots to be transported to or from the belt conveyor (30). Motor means isprovided for moving the belt conveyor (30) continuously. When a belt(26) is moved in its gutter in one direction, the row of pots supportedon that belt is moved towards a transfer station at which an endmost potof the row is transferred to the belt conveyor (30). When moved in theother direction the belt (26) moves the row of pots supported on thatbelt away from the belt conveyor (30), allowing space for a pot to beintroduced to the end of that row. Each shuttle robot (32, 34) isarranged for movement along the belt conveyor (30) so that it maycommunicate with the gutters individually as desired.

They are of similar construction and comprise guide members (not shown)for guiding pots moving along the belt conveyor (30). A cylinder of apneumatically operated piston and cylinder device (not shown) is mountedon the shuttle robot between the guide members and its piston isarranged for movement horizontally across and above the belt conveyor(30). In its rest position, the piston serves to arrest a pot deliveredfrom a gutter by its belt (26). When it is desired to remove a pot fromthe belt conveyor (30), the piston is actuated to push the pot and urgeit into the selected gutter (22).

Historical positional data combined with fertiliser and watering dataenables an operator of the apparatus to keep track of the nutritionalregime of every single plant in the array. The information also enablesthe operator to schedule all plant movements in the most efficient way.

The apparatus is arranged so that the shuttle robots are actuated inresponse to data contained in the database so as to move a pot from onelocation to another.

1. A process for breeding plants wherein the influence ofenvironment-associated phenotype is reduced compared to conventionalbreeding processes, which process comprises a) growing plants of aspecies in an array of containers charged with growing medium of uniformcharacteristics in an environment of controlled climatic conditions withcontrolled supply of nutrients and feed water wherein the containers aresupported in a horizontally disposed uniform array on a transporterapparatus comprising a plurality of co-extensive storage transportersand a transfer conveyor; b) changing the positions of the containerswithin the environment at intervals during the growing cycle to ensureat least substantially uniform exposure of all plants in the containersto conditions in the environment by moving one or more of the containersfrom one storage transporter to one or more different storagetransporters by a motor via a transfer conveyor located transversely tothe storage transporters, and c) selecting plants for further breedingby comparing the phenotypic characteristics of the plants.
 2. Theprocess according to claim 1, wherein the position of at least onecontainer is changed at intervals of time from six hours to two weeks.3. The process according to claim 1, wherein changes of positions of thecontainers are programmed.
 4. The process according to claim 1, whereinthe position of containers within the environment is changedautomatically.
 5. The process according to claim 1, further comprisingidentifying phenotypic characteristics of the plants at intervals in thegrowing cycle.
 6. The process according to claim 1, wherein the motorassociated with the transporter apparatus is caused to move thecontainers by a predetermined distance.
 7. The process according toclaim 6, wherein a storage transporter is moved by its motor to move therow of containers supported on that storage transporter towards a firsttransfer station at which an endmost container of the row is transferredto a transfer conveyor, the transfer conveyor is operated to move acontainer supported on it to a second transfer station and the motor ofa second storage transporter is operated to collect the container. 8.The process according to claim 7, wherein the container is carried in alinear path from the first mentioned storage transporter to the secondstorage transporter.
 9. The process according to claim 7, wherein thecontainer is carried in a closed path from the first mentioned storagetransporter to the second storage transporter.
 10. The process accordingto claim 1, wherein each container contains a single plant.
 11. Theprocess according to claim 1, wherein said plants are open-pollinatingplants or self-pollinating plants.
 12. The process according to claim 1for the breeding of open pollinating plants in a greenhouse, comprisingthe steps of: a) providing a male sterile open pollinating plant; b)contacting said male sterile plant with pollen; and optionally c)selecting plants having a desired phenotype.
 13. The process accordingto claim 12, wherein said contacting comprises introduction of said malesterile plant into a pollination chamber and the growing of said malesterile plants in said pollination chamber for a period of about 2 daysto about 3 weeks.
 14. The process according to claim 12, wherein saidopen pollinating plant is corn.
 15. The process of claim 1, wherein eachstorage transporter comprises a belt which is moved by means of a motor.16. The process of claim 15, wherein the belt of the storage transportercan be pulled back or forth by the motor allowing the transport ofcontainers to or from the storage transporter.
 17. The process of claim16, wherein the motor is in a shuttle robot which can position itself infront of the storage transporter.
 18. The process of claim 1, whereinthe ends of the storage transporters communicate with the transferconveyor allowing containers to be transported back and forth fromstorage transporter to storage transporter and back and forth tospecific areas in the array.
 19. The process of claim 1, wherein theplants are transgenic plants.
 20. The process of claim 1, wherein theplants are corn or rice plants.
 21. The process of claim 20, wherein thecorn plants are derived from the variety Gaspe.
 22. The process of claim1, wherein the phenotypic characteristic is growth, yield, or stresstolerance.
 23. A process for improved breeding of transgenic plantscompared to conventional breeding, comprising a) growing transgenicplants in an environment of controlled conditions; b) changing theposition of the transgenic plants within the environment automaticallyat intervals during their growing cycle so as to avoid extended exposureto a particular micro-environment, thereby reducing the influence ofmicro-environment variations on the phenotype of the plants; c)comparing phenotype characteristics of the transgenic plants; and d)selecting one or more transgenic plant with a particular phenotype forfurther breeding.
 24. The process of claim 23, wherein the transgenicplants are corn or rice plants.
 25. The process of claim 24, wherein thecorn plants are derived from the variety Gaspe.
 26. The process of claim23, wherein the phenotype characteristic is growth, yield, or stresstolerance.